Brown and colleagues have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor (GPCR) superfamily, specifically subfamily 3 which includes metabotropic glutamate, GABA-B, taste, and putative pheromone receptors. The CaR is expressed in a variety of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells, and is involved in extracellular calcium homeostasis. The CaR cDNA predicts a 7 transmembrane core typical of GPCR but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying the receptor's structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised monoclonal antibodies to synthetic peptides corresponding to sequences in the ECD of the receptor. These antibodies have proved very useful in immunoblot and immunocytochemistry studies of the receptor. We have also succeeded in expressing and purifying the ECD,and in generating monoclonal antibodies against the purified ECD. These antibodies have interesting functional effects on the CaR, and are being evaluated for their epitopes to help define receptor structure/function. Biochemical characterization of the ECD included N-terminal sequencing to define site of signal peptide cleavage, definition of carbohydrate content, secondary structure by CD, and sites of tryptic cleavage. We found that the ECD is an intermolecular disulfide-linked dimer that accounts for the dimeric nature of the intact receptor. Mutagenesis of ECD cysteines has defined which are essential for receptor expression and has identified the cysteines (129 and 131) responsible for receptor dimerization. Disulfide mapping of the ECD using cleavage of the purified protein followed by mass spectrometry is in progress. We have also identified the glycosylation sites critical for receptor expression at the cell surface. We have characterized the functional effects of missense mutations identified in subjects with autosomal dominant hypocalcemia. Most such mutations cause increased sensitivity of the receptor to calcium. We have modeled the ECD structure based on its homology to bacterial periplasmic binding proteins known to have a bilobed, "venus flytrap" structure, and are testing this model using mutagenesis and biochemical approaches .We have shown that a putative loop in the ECD comprising residues ~115-139 forms the dimer interface and is critical for receptor activation. This loop is a "hotspot" for naturally occurring, activating mutations. We have also shown that a cysteine-rich region at the end of the ECD forms a separate domain that plays a critical role in linking calcium activation of the ECD venus flytrap domain to activation of the 7 transmembrane domain of the CaR. We have identified three acidic residues in the second extracellular loop of the 7 transmembrane domain that regulate receptor response to calcium, and a fourth acidic residue in the third extracellular loop critical for the action of a positive allosteric modulator of the receptor. Future studies, including engineered mouse models expressing modified versions of the receptor and/or the G proteins to which it couples, as well as efforts to identify additional allosteric modulators, are directed at elucidating in detail the mechanism of receptor activation by calcium.